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1 International Centre for Genetic Engineering and Biotechnology, Protein Structure and Function Group, 34012 Trieste, Italy
2 Agricultural Biotechnology Center, 2101 Gödöllõ, Hungary
3 Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Reprint requests to: Prof. Sándor Pongor, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Italy; e-mail: pongor{at}icgeb.trieste.it; fax: 39-040-226-555.
(RECEIVED June 4, 2001; FINAL REVISION August 2, 2001; ACCEPTED August 10, 2001)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.21701.
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Abstract |
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 TOP Abstract IntroductionResultsDiscussionMaterials and methodsElectronic supplemental materialReferences |
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Keywords: Protein chemistry; protein structure; proteinase inhibitors; evolution
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsElectronic supplemental materialReferences |
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50-residue-long, cysteine-rich polypeptides that inhibit both trypsin and 
-chymotrypsin. Their three-dimensional structure is characterized by four disulfide bonds, a triple-stranded ß-sheet, and a long loop that interacts with the active site of the proteinase (Greenblatt et al. 1989; Bode and Huber 1992; Nielsen et al. 1994 Nielsen et al. 1995). These proteins are expressed in various tissues of the members of the Solanaceae family, sometimes at very high levels (Graham et al. 1985a; Keil et al. 1986; Johnson and Ryan 1990; Balandin et al. 1995; Miller et al. 2000). Several genes encoding PT-II proteins have been ideied (for a recent overview, see Miller et al. 2000). These genes usually encode a larger precursor protein, which is subsequently cleaved into several slightly different PT-II inhibitor molecules (Table 1
). The precursors contain a variable number of cysteine-rich repeats that we had termed inhibitor precursor (IP)-repeats after the Swiss-Prot name of the parent proteins (Murvai et al. 1999). Proteolytic cleavage of the precursor occurs within, rather than between, repeats (Atkinson et al. 1993). It has been hypothesized that the gene encoding the precursor protein arose from the duplication of a gene originally encoding a single IP-repeat, which was then followed by a frame shift in the proteolytic processing (Scanlon et al. 1999). To support this hypothesis, Scanlon and associates designed and expressed in Escherichia coli an hypothetical ancestral protein corresponding to a single repeat of the Nicotiana alata precursor protein and determined its three-dimensional structure by nuclear magnetic resonance (NMR). This product actually inhibits trypsin and chymotrypsin, and its fold is very similar to that of the naturally occurring inhibitors (Scanlon et al. 1999). In other terms, the N. alata repeat has the ability to fold both as a structural repeat (similar to mature PT-II inhibitors) and as a sequence repeat (similar to aPI1; Scanlon et al. 1999).
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Results |
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-chymotrypsin-Sepharose, and yields two main fractions shown in Figure 1. Mass spectrometry analysis indicated that fraction I contains PSI-1.1, a member of the PT-II family of inhibitors that had been ideied previously (Antcheva et al. 1996). Fraction II contained two products designated as PSI-1.2A and PSI-1.2B. These components were repurified by narrow-bore reverse phase–high-performance liquid chromatography (RP-HPLC) before sequencing (data not shown).
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) consisted of two overlapping peaks. Initial sequencing attempts of the larger peak (A) failed to detect a sequenceable N terminus. A sample was thus reduced, pyridylethylated, and digested separately with either CNBr in 70% HCOOH or trypsin. The resulting peptides (Fig. 2, PSI-1.2A-F1 and PSI-1.2A-F2, respectively) were isolated by narrow-bore RP-HPLC and sequenced. The smaller peak (B), on the other hand, gave a full sequence of 52 amino acids, identical with that of peak A. A comparison of the sequence (Fig. 2) and the observed molecular mass (Fig. 1) indicates that the difference between peak A and peak B results from an unideied N-terminal modification of peak A. The PSI-1.2 sequence (Fig. 2) has eight cysteines, same as in the previously isolated PSI-1.1 (Antcheva et al. 1996).
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). The Q9SDL4 precursor can, in principle, yield three mature PT-II proteins. Interestingly, PSI-1.1 is identical with the first putative cleavage product.
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). This analysis in itself did not yield the complete disulfide topology of the protein. In particular, the connectivity of two adjacent cysteines, Cys31 and Cys32, is not unambiguous (see inset in Fig. 4). Additional data were collected by Edman degradation combined with phenylhydantoin (PTH) analysis at 313 nm, which makes it possible to idey PTH-dehydroalanine (PTH-DHA), the ß-elimination product of PTH-cystine. PTH-DHA forms when the process of N-terminal sequencing reaches a Cys residue that is disulfide bonded to a sequentially upstream Cys residue (Li and Liang 1999). This analysis showed PTH-DHA at positions 28, 32, 38, and 49; an example is shown in Figure 4. These results, combined with the data of Table 2, confirmed that PSI-1.2 has the disulfide topology Cys3-Cys32, Cys7-Cys28, Cys16-Cys38, Cys31-Cys49. The disulfide bridges of PSI-1.2 thus correspond to those of the aPI1 hypothetical ancestral protein (Scanlon et al. 1999).
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-chymotrypsin (Ki = 1.1 x 10-8 M), whereas elastase and subtilisin DY are not inhibited (Table 3). The enzymes thrombin and factor Xa, related to the blood clotting system, are only weakly inhibited by PSI-1.2 (Ki = 1.1 x 10-6 M and Ki = 2.6 x 10-5 M, respectively). As a whole, PSI-1.1 appears to be a stronger inhibitor of thrombin (100x), trypsin (10x), and factor Xa (10x) than the presently isolated PSI-1.2. Pepsin was found not to hydrolyze PSI-1.2 over a period of 30 min at pH 2.0. Heat treatment (100°C) at pH 4.0 for 10 min had no effect on the anti-trypsin activity of PSI-1.2B.
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), the repeat unit of the PT-II family precursors (Murvai et al. 1999). A comparison with mature PT-II inhibitors reveals, on the other hand, that the sequence of PSI-1.2 is circularly permuted compared with that of the mature proteins, as if a domain swapping event had taken place (Bennett et al. 1995; Heringa and Taylor 1997). For example, of the 45 residues of the potato tuber inhibitor PCI-1 (PDB code 4SGB_I) that can be aligned with PSI-1.2, 25 are identical (56%). If we divide PSI-1.2 into three fragments, A-P-B, PCI-1 should be represented as B-A, with P denoting the putative processing site, which is missing in the mature PCI-1 protein (Fig. 3B).
A database search for IP-repeats similar to PSI-1.2 in databases resulted in the ideication of several proteinase precursor proteins containing from two to eight IP-repeats (Table 1). In the precursor proteins from Nicotiana (tobacco), Solanum (potato), and Lycopersicon (tomato), the number of IP-repeats can vary from two to eight. In general, the precursor consists of an integer number of IP-repeats, the only exception being IP2X_SOLTU, for which a short C-terminal partial repeat containing two cysteines is present. The C. annuum precursors IP22_CAPAN and Q9SDL4 are unique in the sense that they contain two IP-repeats flanked by N- and C-terminal partial repeats. Their N-terminal partial repeat contains six cysteines, whereas the C-terminal partial repeat contains three cysteines only. In several cases, different IP-repeats share the same amino acid sequence. For example, five of the Q9SDW7 repeats (R2 to R6) are 100% identical to R4 through R8 from Q9SDW8. The previously isolated PSI-1.1 (IP21_CAPAN) protein (Antcheva et al. 1996) is identical to residues 36–90 of Q9SDL4, indicating that it derives from the proteolytic processing of the Q9SDL4 precursor.
The exon/intron structure was reported for a number of genes. In the known cases, all the IP-repeats (i.e., the entire precursor apart from the signal peptide) are coded by one single exon.
We performed a systematic sequence comparison on the individual IP-repeats found in the databases. For better ideication, the repeats are numbered as R1 through R8, starting from the N terminus of the precursors, and the precursors are denoted by the number of IP-repeats indicated in Table 1. For example, a precursor with two IP-repeats is denoted IP-2. The multiple alignment (deposited) revealed that only the cysteine pattern is completely conserved. The results of the comparison are displayed as a phylogenetic tree (Fig. 5) as well as in a graphical form (Fig. 6). One major cluster includes R1 and R2 repeats from precursors with two and three IP-repeats (IP-2 and IP-3), including IP2X_SOLTU, which has an additional short truncated repeat at the C terminus (IP-2+). This group includes all the known potato precursors as well as the one expressed in tobacco leaves. The second group contains all the IP-repeats of the N. alata and N. glutinosa precursors that consist of four to eight IP-repeats (IP-4, IP-6, and IP-8). These precursors are expressed in floral organs and young tissues of the plants.
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).
Model building
To idey the putative region responsible for proteinase inhibition, we built a three-dimensional model of PSI-1.2 based on the NMR structure of the proteinase inhibitor aPI1 (Scanlon et al. 1999). PSI-1.2 and aPI1 have a 53% sequence identity and the same disulfide bond pattern, so they are likely to adopt the same fold. Figure 7 compares the aPI1-based model of PSI-1.2 with the crystallographic structure of the potato chymotrypsin inhibitor-1 (PCI-1) complexed with the proteinase B from Streptomyces griseus (PDB code 4SGB). Of the available proteinase/inhibitor complexes, 4SGB_I shows the highest sequence similarity to PSI-1.2. Figure 7 clearly shows that despite the different positions of the N and C termini, the overall fold of the two proteins and the orientation of the disulfide bonds are very similar, with a conserved core composed of a triple-stranded anti-parallel ß-sheet and a network of four disulfide bridges. The PCI-1 residues involved in contacts with the active site of the S. griseus proteinase are located in the long loop connecting strands 2 and 3. These residues (33-PKACPLNCDP-42) correspond to the N-terminal region in PSI-1.2. The central eight residues of PSI-1.2 (34-KACPRNCD-41) are well conserved, with the only exception of the L
R substitution. Although constrained by two disulfide bridges at positions 3 and 7, this region shows a high root mean square derived value in the NMR structure of aPI1 and is thus likely to be conformationally flexible.
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, inset). In the hypothetical helix, residues S, E, R, R, and K are facing the solvent, whereas residues G, I, I, V, and C face the interior of the protein. This asymmetric distribution of hydrophilic and hydrophobic residues is unique to PSI-1.2 among the known IP-repeats. |
Discussion |
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). Homology modeling confirmed that the sequence can be built into the structure of aPI1 without steric clashes. In the region that corresponds to the in vivo proteolytic processing site in the aPI1 sequence, PSI-1.2 has the theoretical capability to form an amphiphilic helix that would have the hydrophobic face toward the interior of the protein and the hydrophilic face toward the solvent. As no other IP-repeat sequence shows similar distribution of hydrophobic and hydrophilic amino acids in the corresponding region, we speculate that this feature may be of significance and worth of structural studies. For example, the amphiphilic helix might contribute to the stability of PSI-1.2 in solution; moreover, it might provide protection against proteolytic cleavage as it might not be recognized by the proteases in the plant secretory pathway (Heath et al. 1995).
The similarities that exist between the individual IP-repeats of the various PT-II precursors (indicated by gray lines in Fig. 5) reveal a consistent pattern that suggests that insertion of double repeats may have taken place several times during the evolution of this protein family. For example, an insertion of two repeats between R3 and R4 of the N. alata precursor Q9SQ77 may have given rise to Q40378. In a similar way, the structure of the eight-repeat precursor Q9SDW8 can be deduced as a result of an analogous insertion N-terminal to R2 in Q9SDW7. This supposition has an interesting implication regarding the potential origins of PSI-1.2. The sequence of PSI-1.2 is most similar to R3 of the three-membered precursors IP-3. The similarity leads us to speculate that IP-3 proteins may in fact have evolved by insertion of a double repeat into a putative ancestral protein IP-1 (Fig. 8A), and PSI-1.2 might in fact be the product of the ancestral IP-1 precursor as predicted by Scanlon and associates (1999). This hypothetical precursor might then give rise to PSI-1.2 by cleavage of the signal peptides without further proteolytic processing. In other terms, all members of the PT-II family might have emerged via a combination of duplications, insertions, and subsequent or concomitant modification events (e.g., emergence of proteolytic cleavage sites, sequence losses in the case of IP-3` precursors) from the same ancestral sequence, as schematically shown in Figure 8A. This explanation is speculative and awaits experimental confirmation. It is equally possible that PSI-1.2 is derived via an alternative proteolytic processing scheme from an unknown precursor consisting of several IP-repeats (Fig. 8B). Irrespective of the precise evolutionary route, it appears that the general mechanisms producing the permuted precursor genes and protein products in this family of Solanaceae genes are presumably similar to those invoked for the explanation of circular rearrangements in other species (Heringa and Taylor 1997; Lindqvist and Schneider 1997; Jeltsch 1999).
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) may have had an interesting implication specific for this family of proteins. The ancestral repeat unit must have formed intra-repeat disulfides only, such as those in aPI1 and in PSI-1.2. In other terms, the ancestral repeat must have been an independent folding unit—as indirectly proved by Scanlon et al. (1999). In contrast, those repeats that are cleaved in their central sections form inter-repeat disulfides (e.g., PCI-1), so that the folding units are now formed from segments of two adjacent sequence repeats. The two disulfide topologies are different, as shown in Figure 7, even though the positions and the spatial orientation of the disulfide bridges are in good agreement. The emergence of an alternative folding ability, followed by the formation of a new disulfide topology, might have been a prerequisite for the new proteolytic processing to appear. We can conclude that PSI-1.2 represents a novel type of plant serine proteinase inhibitor, which is related to the mature PT-II inhibitors by a circular permutation in the amino acid sequence. It corresponds to a complete IP-repeat unit of the precursor proteins and is derived from a hitherto unideied member of PT-II precursors. In the members of this protein family present in Solanaceae, rearrangements have apparently taken place both at the DNA level (duplications, insertions, loss of flanking sequences) as well as on the protein level (proteolytical processing variants, single-chain versus two-chain analogs). Nevertheless, the resulting protein products all seem to fold into the same overall three-dimensional structure, which may be required for binding to proteinases. The case of PSI-1.2 is apparently the first example in which circularly permuted members of a protein family are expressed within the same organism.
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Materials and methods |
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-chymotrypsin, elastase, and human plasma thrombin were purchased from Sigma; human plasma coagulation factor Xa, from Calbiochem; and pepsin, from Merck. Subtilisin DY was isolated according to Nedkov and Bobatinov (1976). H-D-Phe-Pec-Arg-pNA•AcOH was purchased from Bachem; N-methoxycarbonyl-Nle-Gly-Arg-pNA•AcOH (Chromozym X), from Boehringer Mannheim; Suc-Ala-Ala-Pro-Phe-pNA (Suc-X-pNA) and N-benzoyl-L-arginine-pNA hydrochloride (L-BApNA), from Fluka; Casein Hammarsten Servabacter, from Serva; and p-nitrophenyl-p`-guanidino-benzoate hydrochloride (p-NPGB) and N-trans-cinnamoyl-imidazole (NTCI), from Sigma.
Inhibitor isolation
The isolation of the inhibitors was performed essentially as described previously (Antcheva et al. 1996). The final purification was performed by RP-HPLC on a semi-preparative Delta Pak 300 15RP18 column (250 x 10 mm) using a Millipore Waters HPLC system. The two solvents used were 0.1% (v/v) TFA in water (solvent A) and 0.1% (v/v) trifluoroacetic acid in acetonitrile (solvent B). A linear gradient was used for elution (flow rate, 6.0 mL/min) in which the solvent composition changed from 0% to 20% solvent B in 45 min. The purity of the isolated proteins was checked on an analytical HPLC column (LiChrosphere 5RP18, 4 x 250 mm, Merck); chromatography was performed at a flow rate of 1 mL/min and gradient from 0% to 45% solvent B in 40 min, using the same buffer system as for the preparative runs. The molecular weight of inhibitors were determined by electron spray mass spectrometry on a Perkin Elmer Sciex API 150EX.
Enzyme inhibition assays
Inhibition of serine proteinases (except subtilisin) were followed by monitoring the rate of hydrolysis of p-NA substrates spectrophotometrically at 405 nm using a Ultrospec 3000 UV/Visible spectrophotometer (Pharmacia Biotech) using L-BApNA (trypsin), Suc-Ala-Ala-Pro-Phe-pNA (-chymotrypsin), Suc-(Ala)3-pNA (elastase), H-D-Phe-Pec-Arg-pNA•AcOH (thrombin), and N-methoxycarbonyl-Nle-Gly-Arg-pNA•AcOH (factor Xa). Precise concentrations of the active enzymes were determined by standard active site titration procedures (Chase and Shaw 1970). In the inhibition assays, stoichiometric concentrations in the10-7 M range of the enzymes (trypsin, -chymotrypsin) and the inhibitors (PSI-1.1 and PSI-1.2) were used. The residual enzyme activity was measured after 5-min incubation of the enzymes with the inhibitors at 22°C in 50 mM Tris-HCl buffer at pH 7.8), containing 0.02 M CaCl2. The substrates (10-4M) were added, and the reaction was followed at 405 nm (Geiger and Fritz 1984). In the inhibition assays of human plasma thrombin and blood coagulation factor Xa, concentrations in the 10-9 M range of the enzymes, 10-7M of the inhibitors, and 10-4 M of the substrates were used. The measurements were performed in 50 mM Tris/HCl, 100 mM NaCl, 0.5 mg/mL BSA, 20 mM EDTA at pH 7.8 and 22°C (van Dam-Mieras et al. 1984). PSI-1.2 was assayed for possible inhibitory activity against subtilisin DY using 1.2% casein as a substrate. The dissociation constants (Ki) of the enzyme-inhibitor complexes were determined by the method of Cha (1975).
Protein sequence determination
Reduced and pyridylethylated samples (Hampton et al. 1992) were digested separately either with CNBr in 70% HCOOH overnight at room temperature or with trypsin for 3 h at 37°C (E:S, 1:20). The resulting peptides were isolated by narrow-bore RP-HPLC on an analytical Aquapore OD300 column (220 x 2.1 mm, 7-mm particle size, Applied Biosystems) using gradients of acetonitrile in 0.1% (v/v) aqueous TFA. The pyridylethylated protein and the peptides were sequenced by automated Edman degradation using an Applied Biosystems protein sequencer (model 471 A) equipped with a 120A PTH analyzer (Hunkapiller et al. 1983).
Determination of disulfide bridges
The disulfide topology of PSI-1.2B was deduced from mass spectrometric and N-terminal sequencing analysis of enzymatic digests of the protein using a combination of methods described previously (Chagolla-Lopez et al. 1994; Lu et al. 1999). Peptide samples corresponding to 75 mu;g PSI-1.2B were dissolved in 150 mu;L of 0.2M Tris-HCl buffer at pH 7.3 and digested with a mixture of trypsin (3mu;g) and chymotrypsin (3mu;g) for 15 h at 37°C. The mixture was separated by RP-HPLC on a Vydac C18 column (2.1 x 250mm) and subjected to mass spectrometry analysis, and the molecular weights were compared with those predicted for the expected peptides. Selected peaks were collected, liophylised, and subjected to automated Edman degradation. The released amino acid phenylthiohydantoins were detected at 269 and 313 nm (Li and Liang 1999).
Sequence similarity searches
The sequence homology search in the SWISS-PROT, trEMBL (Bairoch and Apweiler 2000), SBASE (Murvai et al. 2000), and PIR (Barker et al. 2000) protein databases for proteinase inhibitor precursor proteins was performed using BLAST (Altschul et al. 1997), FASTA (Pearson and Lipman 1988), and BLITZ (Collins and Coulson 1990). Full-length precursors only were retained. The precursors were dissected into repeats homologous to the PSI-1.2 sequence and clustered using CLUSTALW (Thompson et al. 1994). The phylogenetic tree was built using the PHYLIP package and drawn using DRAWGRAM according to Felsenstein (1981) using a neighbor-joining algorithm (Saitou and Nei 1987).
Modeling
The three-dimensional model of PSI-1.2 was built from the NMR structure of the N. alata aPI1 putative inhibitor. PSI-1.2 and aPI1 were aligned manually, and the first five structures of the NMR ensemble (PDB code 1CE3) were used as templates. The model was built by satisfaction of spatial restraints and energy minimization using MODELLER (Sali and Blundell 1993). The molecules were displayed using MOLMOL (Koradi et al. 1996).
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Electronic supplemental material |
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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